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New Insights into the Cytotoxic Mechanism of Hexabromocyclododecane (HBCD) from a Metabolomic Approach Feidi Wang, Haijun Zhang, Ningbo Geng, Baoqin Zhang, Xiaoqian Ren, and Jiping Chen Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03678 • Publication Date (Web): 13 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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Environmental Science & Technology

1

New

Insights

into

the

Cytotoxic

Mechanism

2

Hexabromocyclododecane (HBCD) from a Metabolomic Approach

3

Feidi Wang,†,‡ Haijun Zhang,†,* Ningbo Geng,†,‡ Baoqin Zhang,† Xiaoqian

4

Ren,†,‡ Jiping Chen†,*

5

†

6

Chinese Academy of Sciences, Dalian, 116023, China

7

‡

Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics,

University of Chinese Academy of Sciences, Beijing 100049, China

8 9 10 11 12

of

TOC/Abstract Art

13 14 15 16 17 18

*Corresponding Authors

19

Phone: +86-411-8437-9972, fax: +86-411-8437-9562; e-mail: [email protected].

20

Phone/fax: +86-411-8437-9562; e-mail: [email protected].

ACS Paragon Plus Environment


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ABSTRACT

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The toxic effects of hexabromocyclododecane (HBCD) are complex, and the underlying

23

toxicological mechanisms are still not completely understood. In this study, a

24

pseudo-targeted metabolomic approach based on the UHPLC/Q-Trap MS system was

25

developed to assess the HBCD-intervention-related metabolic alteration in HepG2 cells. In

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addition, some physiologic indicators and relevant enzyme activities were measured. HBCD

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exposure obviously impaired metabolic homeostasis and induced oxidative stress, even at

28

an environmentally relevant dose (0.05 mg/L). Metabolic profiling and multivariate

29

analysis indicated that the main metabolic pathways perturbed by HBCD included amino

30

acid metabolism, protein biosynthesis, fatty acid metabolism and phospholipid metabolism.

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HBCD suppressed the cell uptake of amino acids, mainly through inhibition of the activity

32

of membrane transport protein Na+/K+-ATPase. HBCD down-regulated glycolysis and

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β-oxidation of long-chain fatty acids, causing a large decrease of ATP production. As a

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result, the across-membrane transport of amino acids was further inhibited. Meanwhile,

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HBCD induced a significant increase of total phospholipids, mainly through the remodeling

36

of phospholipids from the increased free fatty acids. The obtained metabolomic results also

37

provided some new evidence and clues regarding the toxicological mechanisms of HBCD

38

that contribute to obesity, diabetes, nervous system damage, and developmental disorders.

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INTRODUCTION

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Hexabromocyclododecane (HBCD) is a brominated cyclic alkane used primarily as an

41

additive flame retardant in polystyrene-based materials including resins and fabrics.1, 2 It

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has been listed as a new persistent organic pollutant (POP) in Annex A of the Stockholm

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Convention in 2013,3 but with specific exemptions for expanded and extruded polystyrene

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foams in buildings until 2024.4 Because of its persistence and lipophilicity, HBCD can

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accumulate in the human body through a combination of diet, dust ingestion and indoor air

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inhalation.5 As a result, HBCD has been frequently detected in human blood and breast ACS Paragon Plus Environment

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milk.6, 7

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The acute toxicity of HBCD appears to be low,5 and it lacks significant genotoxic

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potential to an organism.1, 8 However, the subacute and subchronic effects of HBCD are

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manifest.9, 10 Hepatic toxicity and thyroid toxicity of HBCD were universally observed in in

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vivo animal studies.11,

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hyperplasia,14 cytochrome P450 enzyme induction,11 and thyroid hormone disruption.12

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Furthermore, potential reproductive effects,15 nervous system damage16 and developmental

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toxicity17 were also observed in response to HBCD exposure.

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The adverse effects include liver weight increase,13 thyroid

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Accumulating evidence shows that HBCD can induce endocrine disruption,13 metabolic

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dysfunction and obesity,18 even at environmentally relevant doses. HBCD has the ability to

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interact with the constitutive androstane receptor (CAR) and pregnane X receptor (PXR).19

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An in vivo study indicated that oral exposure to HBCD gave rise to a dose-dependent

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decrease of trabecular bone mineral density in female rats with a BenchMark Dose Lower

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confidence bound (BMDL) of 0.056 mg/kg body weight/day.20 Another in vivo study found

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that the body and liver weight were markedly increased in high-fat diet mice treated with

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HBCD at a medium dose of 35 and a high dose of 700 µg/kg body weight/week, paralleled

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by the disruption of lipid and glucose homeostasis.18 These low-dose effects raise serious

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concerns for human health based on human body burdens of HBCD, particularly for the

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occupational exposure setting.

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In the past decade, significant advances have been made toward understanding

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mechanisms underlying the adverse effects of HBCD. As a xenobiotic, HBCD is first

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metabolized by cytochrome P450 enzymes, and thereby reactive oxygen species (ROS) are

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generated.21 Overproduction of ROS likely results in oxidative damage to lipids, proteins

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and DNA.21 Meanwhile, HBCD-induced activation of the PI3K/Akt pathway and

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Nrf2-ARE pathway provide a cytoprotective process that responds to HBCD-induced ROS

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generation.22, 23 HBCD can induce CYP2B and CYP3A enzymes through interaction with ACS Paragon Plus Environment


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CAR/PXR receptors, which may account for the disruption of the thyroid hormone axis.19

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Several in vitro studies also indicated the inhibitory effect of HBCD on the

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sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, which is accompanied by an

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intracellular Ca2+ increase and mitochondrial dysfunction.24, 25 Gene expression profiles in

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rat livers revealed that HBCD exposure disrupted several specific pathways, such as

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PPAR-mediated regulation of lipid metabolism, triacylglycerol metabolism, cholesterol

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biosynthesis, and phase I and II pathways.9 Furthermore, untargeted metabolomics based on

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in vitro study revealed that exposure to HBCD for 72 h could significantly induce the

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metabolic changes.26 However, the metabolic pathways intervened by HBCD exposure

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require further confirmation, and there still remains a lack of metabolomic evidence

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supporting the underlying mechanisms of HBCD toxicity.

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The biological events induced by HBCD appear to be complex, and the underlying

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toxicological mechanisms of HBCD are not completely elucidated. To better understand the

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mode of action of HBCD, metabolomic evidence is required. Metabolomics aiming to

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systematically study small molecule metabolites is a method of understanding metabolic

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regulation.27 It can offer a perspective on how mechanistic biochemistry relates to the

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phenotypic state of an organism because metabolites serve as direct signatures of

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biochemical activity.28 The intrinsic physiological responses of an organism after HBCD

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exposure are reflected and propagated by its metabolism, and conversely, the variation in

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metabolic fingerprints can reveal and verify the mechanisms that underlie the toxicity of

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HBCD.29 In this study, the cytological effects of HBCD exposure on HepG2 cells were

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examined, and a pseudo-targeted metabolomic analysis was performed to investigate

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HBCD-intervention-related metabolic alteration. The obtained results are expected to

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provide a better understanding of metabolic disturbances induced by HBCD exposure and

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provide new evidence and clues concerning the toxicological mechanisms of HBCD from a

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metabolomic perspective. ACS Paragon Plus Environment

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EXPERIMENTAL SECTION

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Cell Culture, Exposure Conditions and Sample Collection. Human hepatoma

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HepG2 cell was adopted as an in vitro model for the study of cytotoxic mechanism. HepG2

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cell has high stability and unlimited life-span, and it retains a wide range of human

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liver-specific functions.30 In addition, its molecular expression and biological phenotypes

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have been extensively characterized.30 The test HepG2 cells were provided by China

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Infrastructure of Cell Line Resources (Shanghai, China), and these cells were maintained in

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Dulbecco’s Modified Eagle Medium (DMEM basic (1X), Gibco-BRL) supplemented with

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10% fetal bovine serum (FBS, Gibco-BRL) and 1% Penicillin–Streptomycin (Beyotime,

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China) under humidified air containing 5% CO2 at 37 °C. Cells in the logarithmic phase of

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growth were rinsed with phosphate-buffered saline, trypsinized, and then seeded in culture

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plates for exposure experiments.

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HepG2 cells were exposed to a reagent-grade HBCD formula (purity: > 95%, Aladdin

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Industrial Corp.) with varying concentrations in the culture medium. HBCD stock solutions

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were made up in ACS grade dimethyl sulfoxide (DMSO; Amresco, Solon, OH, USA).

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After cells were seeded and grown to 80% confluency, HBCD stock solution was

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incorporated into the cell culture medium. The final DMSO content in the culture medium

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was 0.05% (v/v), and background control cells were treated with only 0.05% DMSO.

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Cell viability was first tested using a thiazolyl blue tetrazolium bromide (MTT, Amresco,

118

Solon, OH, USA) assay to ascertain the appropriate exposure conditions for metabolomic

119

study. HepG2 cells were exposed to a series of HBCD concentrations with incubation

120

durations of 1, 2 and 3 days. The details of the cell viability assays are shown in the

121

Supporting Information. According to the results of MTT assay (see Supporting

122

Information), an exposure time of 1 day was adopted for metabolomic study; and three

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HBCD exposure doses relating to different effects on cell viability, the lowest dose with

124

observable inhibition effect (0.05 mg/L), a middle dose without observable effect (1 mg/L) ACS Paragon Plus Environment


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and the highest dose with observable stimulation effect (10 mg/L), were selected. The

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exposure dose of 0.05 mg/L is comparable with the maximum serum concentration of

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HBCD (856 ng/g lipid) in individuals occupationally exposed.7

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In the metabolomic study, HepG2 cells were seeded in 6-well plates at about 3 × 105

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cells/well and incubated with HBCD at concentrations of 0.05, 1 and 10 mg/L for 24 h.

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After exposure, 200 µL of culture medium was transferred to an Eppendorf tube for

131

analysis of extracellular metabolites, and then the remaining culture medium was removed.

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Cells were rinsed by gently dispensing ultrapure water to the surface twice, aspirating

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rapidly, and then quenched by liquid nitrogen freezing. The quenched cells adhering to

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plates, together with the culture medium in Eppendorf tubes, were stored at –80 °C and

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then extracted within 7 days.

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Metabolomic Analysis. To analyze the intracellular metabolites, 1 mL of ultrapure

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water was added to each well, followed by ultrasonic disruption in an ice-water bath for 3

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min, and then the suspension containing cell fragments was transferred into an Eppendorf

139

tube. The freeze-drying of the samples was then performed simultaneously for the disrupted

140

cells containing intracellular metabolites and collected culture medium containing

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extracellular metabolites. Soon afterwards, metabolites in the freeze-dried samples were

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dissolved in 0.5 mL of 80% methanol, vortexed for 20 min, and centrifuged for 20 min at

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13,000 × g and 8 °C. Finally, the supernatant was filtered by an organic phase filter and

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transferred to a vial for metabolite analysis. Six internal standards (L-phenylalanine-d5,

145

octanoyl

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1,2-diheptadecanoyl-sn-glycero-3-phosphoethanolamine,

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nonadecanoic acid) served as quality controls for sample preparation and instrumental

148

analysis.

(8,8,8-D3)-L-carnitine,

1-lauroyl-2-hydroxy-sn-glycero-3-phosphocholine, hendecanoic

acid

and

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Metabolomic analysis adopted a pseudo-targeted approach, which can display better

150

repeatability and wider linear range than the traditional Q-TOF MS-based untargeted ACS Paragon Plus Environment

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metabolomic method.27, 31 In brief, the extracts from HepG2 cells and culture medium were

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analyzed by a Waters Acquity Ultra Performance liquid chromatography coupled online to

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an ABI Q-Trap 5500 (AB SCIEX, USA) system (UHPLC/Q-Trap MS) operated in multiple

154

reaction monitoring (MRM) mode, for which the MRM ion pairs were acquired from the

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cell extracts through untargeted tandem MS using an Agilent 1290 Infinity Ultra

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Performance liquid chromatography system coupled online to an Agilent 6540 UHD

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Q-TOF MS (Agilent, Santa Clara, CA) system (UHPLC/ Q-TOF MS). The same condition

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of liquid chromatography was performed on both UHPLC/Q-Trap MS and UHPLC/Q-TOF

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MS. A total of 241 ion pairs with defined parameters were analyzed in the mode of positive

160

electrospray ionization (ESI+) with an Acquity UPLC BEH C8 column (2.1 mm × 100 mm,

161

1.7 µm, Waters, USA). In ESI– mode, an Acquity UPLC HSS T3 column (2.1 mm × 100

162

mm, 1.8 µm, Waters, USA) was used for the chromatographic separation of 59 ion pairs.

163

The details of instrumental analysis are shown in Supporting Information.

164

Sequence analysis of intracellular metabolites and extracellular metabolites were

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performed. To ensure data quality for metabolic profiling, pooled quality control (QC)

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samples were prepared by mixing all of the samples. During analysis of the sample

167

sequence, 7 replicates of the QC samples were inserted into the analytical sequence.

168

Validations of the method, including precision, stability, and recovery, were carried out.

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The results indicated that this metabolomics method is reliable (see Supporting

170

Information).

171

Data Processing. MultiQuant software (3.0.1, AB SCIEX) was used for data processing

172

by analyzing the extracted ion chromatograms of the Q-Trap MS data. After the peak

173

alignment and the removal of the missing values, ion peak areas across ESI+ and ESI–

174

modes were normalized to internal standards and then merged into one data set. The

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merged data set of each sample was further normalized to the sum of peak areas to balance



176

the difference in cell number. A one-way analysis of variance (ANOVA) was performed

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using SPSS PASW Statistics software (SPSS Inc., Chicago, IL), and P values of < 0.05

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were considered as significantly different from the control. Principal component analysis

179

(PCA) and partial least squares discriminate analysis (PLS-DA) were applied with unit

180

variance (UV) scaling using SIMCA-P11.5 software (Umetrics, Sweden), and hierarchical

181

cluster analysis (HCA) was conducted using the MeV software package (version 4.8.1).

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The correlation network was constructed using the Cytoscape software package (version

183

2.8.2). The metabolite set enrichment analysis and pathway analysis were based on

184

MetaboAnalyst, a web service for metabolomics data analysis.

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Determination of Physiologic Indicators and Relevant Enzyme Activities. To

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further understand and verify the results of the metabolomic study, ROS generation, ATP

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level, several oxidative stress markers, together with activities of some crucial enzymes

188

regulating glycolytic pathway, transport across cellular membranes, β-oxidation of fatty

189

acid and phospholipid metabolism, were determined. The selected oxidative stress markers

190

included the levels of reduced glutathione (GSH) and malondialdehyde (MDA) as well as

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the activities of superoxide dismutase (SOD) and catalase (CAT). Intracellular ROS levels

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in HepG2 cells were assayed using a fluorescence microscope with 2,7-dichlorofuorescin

193

diacetate (DCFH-DA), according to the instructions of the ROS detection kit manufacturer

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(Nanjing Jiancheng Bioengineering Institute, China). The specific assay kits (Nanjing

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Jiancheng Bioengineering Institute, China) and enzyme-linked immune sorbent assay

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(ELISA) were adopted to determine the oxidative stress biomarkers and the activities of

197

relevant metabolic enzymes, and the method details are shown in Supporting Information.

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RESULTS AND DISCUSSION

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Basic Physiological Effects Induced by HBCD. Intracellular ROS plays an

200

important role in cell signaling and homeostasis. In this study, HBCD exposure gave rise to


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a linear increase of intracellular ROS with increasing doses, and the increases were

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significant at all three exposure doses (0.05, 1 and 10 mg/L) compared with the control

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group (Figure S3). The over-production of ROS resulted in an observable oxidative stress

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response primarily characterized by the elevated activity of CAT (Figure S4). Moreover,

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exposure to a high-dose of HBCD caused a reduction in SOD activity and an increase of

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GSH levels (Figure S4). The MDA level, as a lipid peroxidation indicator, was also

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significantly altered by HBCD intervention (Figure S4).

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Cell viability was examined based on five exposure doses (0.05, 0.5, 1, 5 and 10 mg/L)

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for 24, 48 and 72 h. As shown in Figure S1, HBCD induced a dose- and time-dependent

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variation of cell viability. After 1 day exposure, cell viability was decreased at a dose of

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0.05 mg/L, whereas the viability of cells in the group with exposure dose of 1 mg/L was

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similar to that of control group. Subsequently, cell viability increased at doses of ≥ 1.0

213

mg/L with a maximum at a dose of 10 mg/L. The increased cell viability was also observed

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for HepG2/C3A cell exposed to HBCD at concentration of 5 µmol/L (equivalent to 3.2

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mg/L) for 1 day.32 Exposure to HBCD at doses of ≤ 5 mg/L for 2 days significantly

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stimulated cell proliferation. However, HBCD produced statistically significant cell

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lethality at all doses after exposure for 3 days. Two previous in vitro studies observed an

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obvious inhibitory effect of HBCD on HepG2 cell at concentrations of 10–100 µmol/L

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(equivalent to 6.4–64.2 mg/L) after exposure for 3 days. 25, 33

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Metabolic Profiling and Multivariate Analysis. A pseudo-targeted metabolomic

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analysis was performed to profile the changes of intracellular metabolites in HepG2 cells

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induced by HBCD intervention. A total of 300 intracellular metabolites were detected.

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Among them, 133 metabolites were identified qualitatively using quasimolecular ions, and

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86 metabolites were further confirmed by authentic standard samples. PLS-DA was

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performed on the normalized data sets for all detected intracellular metabolites. The



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PLS-DA score plot showed a clear separation between HBCD-exposed groups and a

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control group along the component 1 direction (Figure 1). Meanwhile, three

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HBCD-exposed groups were clearly separated from each other along the component 2

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direction. This result indicated that HBCD caused a significant metabolic perturbation.

230 231 232 233 234

Component 2 (14.3%)

20

10

10 mg/L 0

control 1 mg/L -10

0.05 mg/L -20 -20

-15

-10 -5 0 5 Component 1 (32.8%)

10

15

235 236

Figure 1. PLS-DA score plot of metabolites in HepG2 cells exposed to HBCD at various doses

237

for 24 h. R2 = 0.977, Q2 = 0.920.

238 239

A one-way ANOVA was performed to find the differential metabolites among different

240

treatment settings. A total of 115 intracellular metabolites showed significant changes (P
0.76 were identified quantitatively and

243

are listed in Table S1. Subsequently, hierarchical clustering was used to arrange these

244

identified metabolites based on their relative contents across groups. A heat map showed

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five dose-response trajectories (Figure S6). Compared with the control, almost all amino

246

acids presented a significant decrease in all three HBCD-exposed groups; whereas, most of

247

the phospholipids and fatty acids displayed a significant increase in low- and middle-dose

248

groups.

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Correlation Network of Differential Metabolites and Disturbance of Metabolic

250

Pathway. The involved pathways of each differential intracellular metabolite were


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ascertained

252

http://www.genome.jp/kegg/), and the results are listed in Table S2. To investigate the

253

latent relationships among the differential metabolites, a correlation network diagram was

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constructed according to a comprehensive pair-wise computation of Pearson correlations

255

between the differential metabolites involved in the same metabolic pathway. As shown in

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Figure 2, the differential metabolites were mainly bridged by fatty acids. All phospholipids,

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including phosphatidyl choline (PC), lysophosphatidyl choline (LysoPC), phosphatidyl

258

ethanolamine (PE), lysophosphatidyl ethanolamine (LysoPE), sphingomyelin (SM) and

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lysosphingomyelin (LysoSM), were highly up-regulated by HBCD exposure either at low-

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and middle-doses or at a high dose. The similar up-regulation was also observed for fatty

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acids. However, the levels of most amino acids were highly down-regulated by low- and

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middle-dose HBCD. Unlike long-chain acylcarnitine (C18:0) and medium-chain

263

acylcarnitine (C8:0), short-chain acylcarnitine (SC-AC, C2–6) presented a high

264

down-regulation at a high dose of HBCD.

by

an

online

database

of

metabolic

pathways

(KEGG,

265

Metabolic pathway perturbation induced by HBCD intervention was further studied

266

using MetaboAnalyst on the basis of the significantly different intracelluar metabolites.

267

Pathway analysis was conducted to reveal the most relevant pathways influenced by

268

different exposure

269

(www.hmdb.ca/) ID that changed significantly upon HBCD intervention, considered to be

270

highly responsible for the perturbation of amino acid metabolism (Figure S7). The most

271

relevant pathways influenced by HBCD were Alanine, aspartate and glutamate metabolism;

272

glycine, serine and threonine metabolism; and arginine and proline metabolism.

273

Furthermore, enrichment analysis indicated that protein biosynthesis, the urea cycle,

274

pantothenate and COA biosynthesis, glutathione metabolism and ammonia recycling were

275

also disturbed (Figure S8).

doses. There were 88 metabolites with identified


HMDB


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277 278

Figure 2. Metabolic correlation networks of the differential metabolites and related pathways.

279

FA:

280

lysosphingomyelin; PC: phosphatidyl choline; LysoPC: lysophosphatidyl choline; PE:

281

phosphatidyl ethanolamine; LysoPE: lysophosphatidyl ethanolamine.

fatty

acid;

SC-AC:

short

chain

acylcarnitine;

SM:

sphingomyelin;

LysoSM:

282 283

Perturbation in Amino Acid Transport and Metabolism. Amino acids are the

284

essential building blocks of proteins, energy sources, metabolite precursors and signaling

285

molecules in all living cells.34 In this study, 15 types of amino acids were supplied in

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DMEM culture medium for HepG2 cell growth. Exposure to HBCD for 1 day did not give

287

rise to a significant variation in the extracellular total contents of amino acids (Figure 3).

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Only histidine, isoleucine, leucine and threonine in culture medium of low- or middle-dose

289

groups showed slight but significant differences from that of control group (Figure S10).

290

However, the intracellular total contents of amino acids in the three exposure groups were

291

all down-regulated significantly (P < 0.01), and the maximum down-regulation (45.2% of

292

control) was observed at a low exposure dose (Figure 3). Except tryptophan in high-dose

293

group, all intracellular amino acid nutrients in three exposure groups were largely and

294

significantly decreased when compared with a control group (Figure S11). These results

295

suggested a possible inhibitory effect of HBCD on amino acid transport.

296

Membrane transport proteins, mainly the SLC-family, mediate the transfer of amino


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acids across cell membranes.34 The transport via most of SLC-family is dependent on the

298

Na+-gradient, which is driven by Na+/K+-ATPase and ATP.34 The activity of

299

Na+/K+-ATPase and ATP levels in HepG2 cells were further determined. As shown in

300

Figure 3, HBCD exposure led to a significant reduction in Na+/K+-ATPase activity in a

301

dose-dependent manner, and the high-dose exposure group showed a reduction of 65.7% in

302

Na+/K+-ATPase activity compared with the control group. This result suggested a direct

303

inhibitory effect. Meanwhile, the levels of ATP were down-regulated significantly by

304

HBCD exposure (Figure 3). The maximum down-regulation (73.8% of control) was

305

observed at a middle exposure dose (Figure 3). Evidently, the significant inhibition of

306

Na+/K+-ATPase activity and the large decrease of ATP levels both caused the suppressed

307

transport of amino acids across the membrane, as indicated by the large down-regulation of

308

intracellular amino acids.

309

A

previous

study

found

that

HBCD

had

the

potential

to

inhibit

the

310

sarcoplasmic-endoplasmic reticulum Ca2+-ATPase by affecting ATP binding and the E2 to

311

E1 transition step.35 In this study, a HBCD-induced decrease of Ca2+-ATPase was also

312

observed (Figure S5). Ca2+-ATPase and Na+/K+-ATPase are both P-type ATPases, which

313

can catalyze the decomposition of ATP into ADP and a free phosphate ion. Therefore, the

314

molecular mechanisms by which HBCD inhibits the Na+/K+-ATPase should be similar to

315

that of Ca2+-ATPase, i.e., altering ATP binding and affecting the E2 to E1 transition step of

316

Na+/K+-ATPase.

317

A map of amino acid-related metabolic pathways was constructed according to KEGG

318

pathways (Figure S9). The levels of all intracellular amino acids were down-regulated

319

significantly after exposure to HBCD at three doses, with the exception of aspartate. The

320

significant down-regulation of amino acid metabolism inevitably affected protein

321

biosynthesis, urea cycle, pantothenate and CoA biosynthesis, glutathione metabolism and

322

ammonia recycling, as indicated by the enrichment analysis (Figure S8). ACS Paragon Plus Environment


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327

40

SLC

100

Na+

80

110

Extracellular glucose

100

328

Na+

Glucose

60 120

50

Amino acids

90

Glucose SGLT +

Na+

Glucose

Na

GLUT

Glucose

Relative abundance (%)

326

Relative content (%)

325

120

Cell outside

324

Amino acids

∑ Extracellular amino acids

Cell intside

140

∑ Intracellular amino acids

30 20

* *

* *

* *

10 0.0015 0.0010

Intracellular glucose

0.0005

330 331 332

150 100 50

Na+/K+-ATPase *

* *

* *

+

Na+

Na

ATP

Na+/K+ -ATPase +

K

0 10 control 0.05 1 HBCD dose (mg/L)

ADP + Pi

Relative content (%)

329

Relative activity (%)

0.00000

150 100 50

ATP * *

* *

* *

0 10 control 0.05 1 HBCD dose (mg/L)

333

Figure 3. Effects of HBCD exposure on the transport of amino acids and glucose across the

334

membrane of HepG2 cells. SLC: amino acid transporter SLC-family; SGLT: glucose transporter

335

SGLT-family; GLUT: glucose transporter GLUT-family. Significant differences were indicated in

336

comparison of the control by T-test. *, P < 0.05; **, P < 0.01.

337 338

Cysteine is an important precursor of cystine, GSH and redox-sensitive proteins, which

339

are necessary antioxidants in cells.36 In this study, the contents of cysteine in three exposure

340

groups were significantly decreased by more than 50% compared with that of a control

341

group. The large down-regulation of cysteine inevitably inhibited the biosynthesis of these

342

antioxidants; otherwise, their content in cells might remarkably increase, responding to the

343

physiological need to reduce the H2O2 to H2O and increase the cellular capability to

344

eliminate the redundant ROS. In addition, the large decrease of neurotransmitters (aspartate,

345

taurine, glycine and alanine) and neurotransmitters-related metabolites (e.g., tryptophan,

346

tyrosine, valine and serotonin) might induce noticeable effects of HBCD on the functioning

347

of the nervous system.

348

Perturbation in Glucose Transport and Glycolysis. The transport of glucose across

349

the plasma membrane is mediated by two groups of glucose transporters: (I) the


Page 15 of 27


350

Na+-dependent glucose transporter SGLT members37 and (II) the Na+-independent glucose

351

transporter GLUT family.38 These two groups of transporters are both partly expressed in

352

the liver.37, 38 Therefore, the HBCD-induced decrease of Na+/K+-ATPase activity would

353

inevitably affect the glucose transport. In this study, the intracellular glucose content in the

354

three exposure groups only presented a slight decrease, but did not show a significant

355

difference from that of the control (Figure 3). Meanwhile, the extracellular contents of

356

glucose in the three exposure groups also showed no significant differences from those of

357

the control group (Figure 3). This implies that the Na+-independent transport mediated by

358

the GLUT family should be predominant. A previous study has indicated that GLUT1 and

359

GLUT9 proteins are the major contributors to glucose influx in HepG2 cells.39

360

The slight decrease of intracellular glucose content might partly result from the

361

glycolysis inhibition produced by HBCD exposure. In order to test the hypothesis, we

362

further determined the activities of two rate-limiting enzymes of glycolysis, hexokinase

363

(HK) and phosphofructokinase (PFK), which catalyze the conversion of glucose to and the

364

conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, respectively. It was found

365

that exposure to a middle-dose or high-dose of HBCD led to significant reductions of

366

49.4% and 82.1%, respectively, in PFK activity when compared with the control group

367

(Figure S5). However, only slight increases of HK activity were induced by exposure to a

368

middle-dose and high-dose of HBCD (Figure S5). On the whole, glycolysis was inhibited.

369

The inhibitory effect can be further verified by the large down-regulation of lactate content

370

(above 45% of control) in the three exposure groups, though the activities of lactate

371

dehydrogenase (LDH) were only slightly changed (Figure S5). The inhibition of glycolysis

372

partly accounted for the large reduction of ATP levels in the HepG2 cells exposed to

373

HBCD (Figure 3).

374

Perturbation in β-oxidation of Free Fatty Acids. The free fatty acids in the



375

cytoplasm are first activated to acylcarnitines before they are carried into the mitochondria

376

and peroxisome where fatty acid β-oxidation occurs.40 As shown in Figure 4, HBCD

377

exposure significantly increased the total content of intracellular long-chain fatty acids

378

(LC-FA, C14–22) by more than 24%, and the total content of LC-AC were also up-regulated

379

significantly in the low- and middle-dose exposure groups. This result clearly indicated an

380

inhibitory effect of HBCD on the metabolism of LC-FA. The activities of two rate-limiting

381

enzymes for β-oxidation, long-chain acyl-CoA synthetase (LCACS) and long-chain

382

acyl-CoA dehydrogenase (LCACD), were further determined. Compared with control

383

group, the activities of LCACD in the three exposure groups were not changed significantly

384

(Figure S5), while the activities of LCACS showed significant decreases of above 45% in

385

three exposure groups (Figure 4). The large reduction in the activity of LCACS could be

386

responsible for the significant suppression of LC-FA β-oxidation. The decreased activity of

387

LCACS could mainly result from the HBCD-induced down-regulation in the expression of

388

genes relevant to LCACS, such as ACSL1, ACSL3, ACSL4 and ACSL5, which has been

389

indicated by the hepatic gene expression profiles in rats exposed to HBCD.9

390

Mitochondria are responsible for the oxidation of the major portion of fatty acids, while

391

very long-chain fatty acids (VLC-FA, C>22) are exclusively oxidized in the peroxisome.41 In

392

this study, the level of intracellular VLC-FA was increased significantly (P < 0.01) only in

393

HepG2 cells exposed to a low-dose of HBCD (Figure 4). Furthermore, MC-AC, as an

394

activated product of medium-chain fatty acids, also showed slightly increased in the three

395

exposure groups compared with the control group (Figure 4). However, the total contents of

396

the SC-AC were largely down-regulated by the low-dose and high-dose HBCD, suggesting

397

an accelerated oxidation of SC-FA (Figure 4). On the whole, the β-oxidation of fatty acids

398

was down-regulated mainly due to the inhibited oxidation of LC-FA, which predominates

399

over free fatty acids in HepG2 cells. The down-regulation of fatty acid β-oxidation is


Page 16 of 27

Page 17 of 27


400

another important reason for the energy deficiency in cells, as indicated by the lower ATP

401

levels.

0.10

VLC-FA

0.08

40

* *

30

0.06

405 406 407

* *

* *

* *

20

0.003 0.002

* *

*

0.001 0.000

LC-acyl-CoA

SC-AC

LC-AC

VLC-acyl--CoA VLC-AC

80 60 40

LC-FA

LCACS

* *

* *

* *

20

SC-AC

LC-acyl-CoA

MC-FA

Cytosol

0.2

VLC-FA

VLC-AC MC-AC VLC-acyl-CoA MC-acyl-CoA

LC-AC

0

O.M.

Inter-membrane space

0.6

0.05

0.4

MC-AC

I.M.

0.04

* *

* *

SC-AC

0.03

SC-acyl-CoA

0.02

409

Peroxisome lumen

SC-acyl-CoA

100 LC- AC

SC-AC

408

VLC-acyl--CoA β-oxidation

0.02

Relative activity (%)

404


0.04

LC-FA

VLCACS

403

b

0.12

MCACS

a

LCACS

402

0.0 control 0.05

1

10

control 0.05

1

LC-AC

MC-AC

LC-acyl-CoA β-oxidation

Mitochondrial matrix

MC-acyl-CoA

10

TAC

Acetyl-CoA

HBCD dose (mg/L)

410

Figure 4. Effects of HBCD exposure on fatty acid (FA) metabolism. a) Relative abundances; b)

411 412

Metabolic pathways. AC: acylcarnitine; VLC: very long-chain; MC: medium-chain; SC:

413

long-chain acyl-CoA synthetase; MCACS: medium-chain acyl-CoA synthetase. O.M.: out

414

membrane; I.M.: inner membrane. Significant differences were indicated in comparison of the

415

control by T-test. *, P < 0.05; **, P < 0.01.

short-chain;

VLCACS:

very

long-chain

acyl-CoA synthetase;

LCACS:

416 417

Perturbation in Phospholipid Metabolism. Phospholipids are a major component of

418

all cell membranes and are also involved in key regulatory functions within cells.42 As

419

shown in Figure 5, the metabolism of intracellular phospholipid was obviously disturbed. In

420

the glycerophospholipid pathway, the major membrane component PC and its hydrolysis

421

product lysoPC were significantly up-regulated at all three exposure doses, and the total

422

contents of lysoPC metabolites, glycerophosphocholine and choline, also showed

423

significant increases in the three exposure groups compared with the control group (Figure

424

5). Moreover, HBCD elevated lysoPE levels at low and middle exposure doses, but PE



Page 18 of 27

425

levels were only slightly increased in the HBCD exposure groups without significant

426

differences when compared to the control group (Figure 5). The slight increase of PE could

427

be attributed to the inhibited biosynthesis of its direct precursor, phosphatidylserine,

428

resulting from the large decrease of L-serine in the cells exposed to HBCD. Furthermore,

429

SM and lysoSM in the sphingolipid pathway were both significantly increased in the cells

430

exposed to a low-dose and middle-dose of HBCD (Figure 5).

433 434

30 20

3.0 LysoPC * * * *

2 * *

10

GP-C

* *

1

Choline (C) * * * *

2.0 1.5

0

14 PC * *

0.8 * *

8

* *

CDP-C

Phospho-C

0.6

GP: Glycerophosphate

PC: Phosphatidyl choline

PS: Phosphatidyl serine

CDP: Cytidine diphosphate

PLA1: Phospholipase A1

PE: Phosphatidyl ethanolamine

PLA2: Phospholipase A2

S1P: Sphingosine-1-phosphate

AA: Arachidonic acid

SM

LysoSM * *

10 * *

* *

8

0.4

6

SM: Sphingomyelin

12

1.0

12 10

2.5

* *

4

1

435 PE

150 Phospho-E

S1P

Sphingosine

0.4

436 0.3

437

CDP-E

1.0

3 LysoPE

2

*

0.8

* *

0.6 GP-E

438

1 control 0.05 1 10 HBCD dose (mg/L)

Ethanolamine (E)

0.4

L-serine

* *

* *

* *

Ceramide

0.2 control 0.05 1 10 HBCD dose (mg/L)

* *

Phospholipids containing AA

PLA1

100 Relative activity (%)

PS

* *

* *

2

6

0.2

0.5

3 * *

PLA2

432


431

50

150

8

PLA2

100

AA

*

6

* *

*

50

4

0 control 0.05 1 10 HBCD dose (mg/L)

control 0.05 1 10 HBCD dose (mg/L)

2

439 440

Figure 5. Metabolic changes of phospholipids after exposure to HBCD. Significant differences

441

were indicated in comparison to the control by T-test. *, P < 0.05; **, P < 0.01.

442 443

Phospholipids can be hydrolyzed by phospholipase A1 (PLA1) and phospholipase A2

444

(PLA2) to form free fatty acids and lysophospholipids, which maintain the homeostasis

445

between phospholipids and free fatty acids. The activity of PLA1 was determined to be

446

almost stable in different treatments, and the activity of PLA2 was only decreased

447

significantly in cells exposed to a high-dose HBCD (Figure 5). These results implied that

448

HBCD seems not to have the potential to disturb the mass balance between phospholipids


Page 19 of 27


449

and free fatty acids at low and middle exposure doses. Therefore, the elevated

450

phospholipids levels in cells should mainly result from the inversion of increased free fatty

451

acids due to the significant inhibition of HBCD on the β-oxidation of fatty acids. A

452

previous in vitro study found that H2O2-induced neuronal cell injury led to the remodeling

453

of phospholipids mediated by PLA2, and the arachidonic acid (AA) level increased prior to

454

the increase in phospholipids.43,

455

phospholipids containing AA were also observed in cells exposed to HBCD (Figure 5). The

456

up-regulation of AA could further inhibit the production of ATP through uncoupling the

457

oxidative phosphorylation in mitochondria.45 In addition, AA is a key inflammatory

458

intermediate. The elavated AA could provide an evidence for the liver weight increase

459

indcued by HBCD exposure, which was observed in in vivo study using Wistar rats.13

460

Proposed Mechanism for HBCD Toxicity. Our data suggest that the basic cytotoxic

461

mechanism of HBCD could be related to an excessive suppression of energy metabolism

462

(Figure 6). First, HBCD adsorbed on the cell membrane directly inhibits the activities of

463

membrane transport protein P-type ATPases, mainly the Na+/K+-ATPase and Ca2+-ATPase.

464

As a result, the cell uptake of amino acids and glucose is suppressed, and the intracellular

465

concentrations of Na+ and Ca2+ would be enhanced. The suppression of amino acid

466

transport across the membrane led to the down-regulation of intracellular amino acids and

467

further inhibits protein biosynthesis. The biosynthesis inhibition of GSH and

468

redox-sensitive protein, resulting from the large down-regulation of intracellular cysteine,

469

will restrict the ability of cells to eliminate the redundant ROS. When HBCD enters into

470

cells, it not only suppresses glycolysis by inhibiting the activity of enzyme PFK, but also

471

decreases the β-oxidation of LC-FA mainly by down-regulating the gene expression

472

relevant to enzyme LCACS.9 The down-regulation of both glycolysis and fatty acid

473

β-oxidation result in a large decrease of ATP production, which further inhibits the

44

In this study, the proportional increases of AA and



Page 20 of 27

474

across-membrane transport of amino acids and glucose. Moreover, the significant inhibitory

475

effect of HBCD on the β-oxidation of LC-FA induces the overall up-regulation of free fatty

476

acids in cells, which further increases the total phospholipids through the remodeling of

477

phospholipids from free fatty acids, mainly mediated by the enzyme PLA2.

478

479 480

Figure 6. Proposed mechanism for HBCD-induced cytotoxicity based on the metabolomic

481

approach. LCACS: long-chain acyl-CoA synthetase; PFK: phosphofructokinase; HK:

482

hexokinase;

483

adenosine-diphosphate; ROS: reactive oxygen species; GSH: reduced glutathione.

LDH:

lactate

dehydrogenase;

ATP:

adenosine-triphosphate;

ADP:

484 485

ASSOCIATED CONTENT

486

Supporting Information

487

Detailed description on cell viability assay, instrumental analysis, the repeatability of

488

metabolic profiling analysis, ROS measurement, oxidative stress marker determination,


Page 21 of 27


489

relevant metabolic enzyme activities and total protein; results of data processing;

490

HBCD-induced perturbation of extracellular and intracellular nutrients.

491

AUTHOR INFORMATION

492

Corresponding Authors

493

* Phone: +86-411-8437-9972, fax: +86-411-8437-9562; e-mail: [email protected].

494

* Phone/fax: +86-411-8437-9562; e-mail: [email protected].

495

Notes

496

The authors declare no competing financial interest

497

ACKNOWLEDGMENTS

498

The authors thank the National Natural Science Foundation of China (Grant No. 21337002)

499

and the National Basic Research Program (Grant No. 2015CB453100) for the financial

500

support.

501

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